Processes that enable the detection or quantification of substances present at very low concentrations in a sample are important tools in many analytical areas. Such processes include methods of measuring low concentrations of analytes in a clinical sample of biological fluids such as urine and blood and of detecting small amounts of drugs and trace residues of chemicals such as pesticides and herbicides in a sample.
In order to be useful, methods of determining low levels of substances in fluid samples must be highly sensitive and accurate. Receptor based assays such as immunoassays exemplify such methods and are currently used to detect substances in very low concentrations in clinical samples of biological fluids such as blood and urine. Immunoassays detect these substances by using an antibody that reacts specifically with the substance to be tested (e.g., a primary antibody).
The detection of antibodies is a useful tool in the diagnosis of diseases caused by antigens. Similarly, detection of autoantibodies is useful in determining a patient's risk of developing a disease. For example, there has been much research relating to detecting autoantibodies as a risk factor for patients developing insulin dependent diabetes mellitus ("IDDM"). There are numerous autoantibodies that are believed to be indicative of IDDM, which is also known as Type I Diabetes or juvenile diabetes. These include insulin autoantibodies, pancreatic islet cell antigen autoantibodies, and most recently autoantibodies to the 65 kd isoform of glutamic acid decarboxylase ("GAD65").
Autoantibodies to GAD65 have been suggested to be one of the earliest markers for the development of IDDM. These autoantibodies are present several years before clinical onset of IDDM, at which time intervention steps could be taken to deter the progression of the disease.
Specific antibodies can only be measured by detecting binding to their antigen or a mimic thereof. Although certain classes of immunoglobulins containing the antibodies of interest may in some cases be separated from the sample prior to the assay (Decker, et al., EP 0,168,689 A2), in all assays, at least some portion of the sample immunoglobulins are contacted with antigen. For example, in assays for specific IgM, a portion of the total IgM can be adsorbed to a surface and the sample removed prior to detection of the specific IgM by contacting with antigen. Binding is then measured by detection of the bound antibody, detection of the bound antigen or detection of the free antigen.
For detection of bound antibody, a labeled anti-human immunoglobulin or labeled antigen is normally allowed to bind antibodies that have been specifically adsorbed from the sample onto a surface coated with the antigen, Bolz, et al., U.S. Pat. No. 4,020,151. Excess reagent is washed away and the label that remains bound to the surface is detected. This is the procedure in the most frequently used assays, or example, for hepatitis and human immunodeficiency virus and for numerous immunohistochemical tests, Nakamura, et al., Arch. Pathol. Lab. Med. 112:869-877 (1988). Although this method is relatively sensitive, it is subject to interference from non-specific binding to the surface by non-specific immunoglobulins that can not be differentiated from the specific immunoglobulins.
Another method of detecting bound antibodies involves combining the sample and a competing labeled antibody, with a support-bound antigen, Schuurs, et al., U.S. Pat. No. 3,654,090. This method has its limitations because antibodies in sera will bind numerous epitopes, making competition inefficient.
For detection of bound antigen, the antigen can be used in excess of the maximum amount of antibody that is present in the sample or in an amount that is less than the amount of antibody. For example, radioimmunoprecipitation ("RIP") assays for GAD autoantibodies have been developed and are currently in use, Atkinson, et al., Lancet 335:1357-1360 (1990). However, attempts to convert this assay to an enzyme linked immunosorbent assay ("ELISA") format have not been successful. The RIP assay is based on precipitation of immunoglobulins in human sera, and led to the development of a radioimmunoassay ("RIA") for GAD autoantibodies. In both the RIP and the RIA, the antigen is added in excess and the bound antigen:antibody complex is precipitated with protein A-Sepharose. The complex is then washed or further separated by electrophoresis and the antigen in the complex is detected. Other methods of detecting bound antigen are described in Masson, et al., U.S. Pat. No. 4,062,935; Soeldner, et al., U.S. Pat. No. 4,855,242; Ito, et al., EP 0,410,893 A2; Cambiaso, et al., U.S. Pat. No. 4,184,849 and Uchida, et al., EP 0,070,527 A1.
There has been much research in the area of evaluating useful markers for determining the risk factor for patients developing diseases, such as IDDM. For example, markers used to detect IDDM include insulin autoantibodies, Soeldner, et al., supra and circulating autoantibodies to glutamic acid decarboxylase ("GAD"), Atkinson, et al., PCT/US89/05570 and Tobin, et al., PCT/US91/06872. In addition, Rabin, et al., U.S. Pat. No. 5,200,318 describes numerous assay formats for the detection of GAD and pancreatic islet cell antigen autoantibodies. GAD autoantibodies are of particular diagnostic importance because they occur in preclinical stages of the disease, which may make therapeutic intervention possible. However, the use of GAD autoantibodies as a diagnostic marker has been impeded by the lack of a convenient, nonisotopic assay.
One assay method involves incubating a support-bound antigen with the sample, then adding a labeled anti-human immunoglobulin. This is the basis for numerous commercially available assay kits for antibodies such as the Syn.sup.elisa kit which assays for autoantibodies to GAD65, and is described in product literature entitled "Syn.sup.elisa GAD II-Antibodies" (Elias USA, Inc.). Substantial dilution of the sample is required because the method is subject to high background signals from adsorption of non-specific human immunoglobulins to the support.
Many of the assays described above involve detection of antibody that becomes bound to an immobilized antigen. This can have an adverse affect on the sensitivity of the assay due to difficulty in distinguishing between specific immunoglobulins and other immunoglobulins in the sample, which bind non-specifically to the immobilized antigen. There is not only a need to develop an assay that avoids non-specific detection of immunoglobulins, but there is also the need for an improved method of detecting antibodies that combines the sensitivity advantage of immunoprecipitation assays with a simplified protocol. Finally, assays that can help evaluate the risk of developing diseases such as IDDM are medically and economically very important. The present invention addresses these needs.
In immunoassays the sequence of binding reactions is usually determined by the sequence of addition of assay specific reagents. Preferred assay protocols minimize the number of assay steps, such as reagent addition, wash steps, and transfers of the assay mixture from one container to another. Early addition of reagents designed to react at late stages of the assay procedure may slow down or prevent required binding interactions. In particular, it is desirable to avoid premature binding to surfaces because reactions at surfaces often suffer from adverse binding kinetics. It would be useful to have a means to carry out the binding steps in an assay mixture that is in contact with a specifically activated surface without premature binding of the resulting immune complex to the surface. It would be useful to eliminate the need to transfer the assay mixture from an inert container in which binding occurs to a surface-activated container where binding of the complex to the surface facilitates its separation.
We have developed a process that simplifies the detection of auto-antibodies such as GAD. U.S. Pat. No. 5,561,049 describes a depletion ELISA procedure (DELISA) for the detection of auto-antibodies. In that invention, the sample is combined with an antigen that binds the antibodies in the sample to form an antigen:antibody complex. In an example of this method, Protein A is used to bind the complex and not bind the antigen when the antigen is not part of the complex. Streptavidin is used to selectively bind the antigen relative to binding the complex when the complex is bound to the Protein A. Protein A can be bound to a soluble polymer or suspendable solid phase. The streptavidin can be bound to a solid phase. Instead of streptavidin, a molecule consisting of two receptors that bind the antigen can be used where each receptor is bound to a signal producing system member. For example, a biotin-GAD conjugate, when combined with a serum sample, binds to any GAD auto-antibody present in the sample. A protein A-dextran conjugate is then added which binds all the immunoglobulins in the sample including the GAD auto-antibody. Upon transfer of this solution to a streptavidin-coated well, only free biotin-GAD conjugate that is not bound to the auto-antibody can bind to the surface. After removing the solution from the well, the free conjugate is detected by measuring the amount of an enzyme labeled anti-GAD antibody that can bind to the surface. In this method the assay mixture must be kept separate from the streptavidin coated surface until the binding reactions are complete in order to avoid detecting conjugate that would otherwise have bound to auto-antibody. It would be useful to have an assay that eliminates the need to transfer the assay mixture to the streptavidin coated surface.
In addition to the use of immunoassays in a clinical setting, immunoassays are useful in other applications. For example, in view of the widespread use of chemicals in the environment, in the form of pesticides and herbicides, there is a need for a simple, quantitative, and accurate method of measuring the levels of these chemicals which may be present in low concentrations in soil, food products, and water samples.
It is therefore desirable to have a method of determining analyte concentrations in fluid samples which is simple and rapid. Such an assay should be useful for any sample that can be homogenized into a fluid medium. It is especially desirable to be able to determine analytes in biological and other samples. It is further desirable to have a method which does not use toxins or expensive reagents. It also highly desirable to have a method that does not require a separation or collection step.
It would be useful to have an immunoassay procedure that is easier and faster to use. It would be useful to have an assay that minimizes the number of transfer steps and permits the binding reactions including binding to the surface to be carried out in the same well.